Characteristics and Young's Modulus of Collagen Fibrils from Expanded Skin Using Anisotropic Controlled Rate Self-Inflating Tissue Expander.

Mechanical properties of expanded skin tissue are different from normal skin, which is dependent mainly on the structural and functional integrity of dermal collagen fibrils. In the present study, mechanical properties and surface topography of both expanded and nonexpanded skin collagen fibrils were evaluated. Anisotropic controlled rate self-inflating tissue expanders were placed beneath the skin of sheep's forelimbs. The tissue expanders gradually increased in height and reached equilibrium in 2 weeks. They were left in situ for another 2 weeks before explantation. Expanded and normal skin samples were surgically harvested from the sheep (n = 5). Young's modulus and surface topography of collagen fibrils were measured using an atomic force microscope. A surface topographic scan showed organized hierarchical structural levels: collagen molecules, fibrils and fibers. No significant difference was detected for the D-banding pattern: 63.5 ± 2.6 nm (normal skin) and 63.7 ± 2.7 nm (expanded skin). Fibrils from expanded tissues consisted of loosely packed collagen fibrils and the width of the fibrils was significantly narrower compared to those from normal skin: 153.9 ± 25.3 and 106.7 ± 28.5 nm, respectively. Young's modulus of the collagen fibrils in the expanded and normal skin was not statistically significant: 46.5 ± 19.4 and 35.2 ± 27.0 MPa, respectively. In conclusion, the anisotropic controlled rate self-inflating tissue expander produced a loosely packed collagen network and the fibrils exhibited similar D-banding characteristics as the control group in a sheep model. However, the fibrils from the expanded skin were significantly narrower. The stiffness of the fibrils from the expanded skin was higher but it was not statistically different.

Improved angiogenic cell penetration in vitro and in vivo in collagen scaffolds with internal channels.

Porous scaffolds are limited in volume due to diffusion constraint and delay of vascular network formation. Channels have the potential to speed up cellular penetration. Their effectiveness in improving angiogenic cell penetration was assessed in vitro and in vivo in 3-D collagen scaffolds. In vitro, channelled and non-channelled scaffolds were seeded with vascular smooth muscle cells. Results demonstrated that the scaffolds supported angiogenic cell ingrowth in culture and the channels improved the depth of cell penetration into the scaffold (P < 0.05). The cells reside mainly around and migrate along the channels. In vivo, channels increased cell migration into the scaffolds (P < 0.05) particularly angiogenic cells (P < 0.05) resulting in a clear branched vascular network of microvessels after 2 weeks in the channelled samples which was not apparent in the non-channelled samples. Channels could aid production of tissue engineered constructs by offering the possibility of rapid blood vessel infiltration into collagen scaffolds.

Biomechanical Effects of Unidirectional Expansion Using Anisotropic Expanders in Horse Skin Tissue.

The use of a self-inflating tissue expander is a technique to stretch cutaneous tissues for potential use in reconstructive skin surgeries. This study investigates the mechanical properties of horse skin stretched by the subcutaneous implantation of anisotropic tissue expanders at the forehead, right shoulder, and dorsomedial part of the cannon region of the right forelimb in six (n = 6) horses. After 14 days of skin expansion, expanded and normal (control) skin samples were harvested and their mechanical properties of elastic modulus (EM), maximum force (MF), maximum stress (MSs) and maximum strain (MSr) were evaluated using uniaxial tension test. The expanded skin from shoulder area has higher EM, MSs, MSr and MF than the normal skin when compared to the forehead and lower forelimb. Statistically, there was a significant (P= .02) mean difference for MSs between the expanded shoulder and lower forelimb skin, but the pairwise comparison of EM, MSr and MF showed no significant difference between the locations. The overall effect of locations on EM and MSs was statistically significant (P < .05), however, there was no overall effect of horse factor, treatment factor (normal and expanded skin) and location interaction on the EM, MSS, MF and MSr. In conclusion, the expanded skin from the frontal head and the distal limb are less elastic (stiffer) compared to that of the expanded skin of the shoulder, thus anatomical location of the skin has some degree of effect on EM and MSs.

Influence of telopeptides, fibrils and crosslinking on physicochemical properties of type I collagen films.

Type I collagen is widely used in various different forms for research and commercial applications. Different forms of collagen may be classified according to their source, extraction method, crosslinking and resultant ultrastructure. In this study, afibrillar and reconstituted fibrillar films, derived from acid soluble and pepsin digested Type I collagen, were analysed using Lateral Force Microscopy (LFM), Fourier Transform Infra-Red Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC) and enzymatic stability assays to asses the influence of telopeptides, fibrils and crosslinking. LFM proved to be a useful technique to confirm an afibrillar/fibrillar ultrastructure and to elucidate fibril diameters. FTIR has proved insensitive to ultrastructural differences involving telopeptides and fibrils. DSC results showed a significant increase in T(d) for crosslinked samples (+22-28 degrees C), and demonstrated that the thermal behaviour of hydrated, afibrillar films is more akin to reconstituted fibrillar films than monomeric solutions. The enzymatic stability assay has provided new evidence to show that afibrillar films of Type I collagen can be significantly more resistant to collagenase (by up to 3.5 times), than reconstituted fibrillar films, as a direct consequence of the different spatial arrangement of collagen molecules. A novel mechanism for this phenomenon is proposed and discussed. Additionally, the presence of telopeptide regions in afibrillar tropocollagen samples has been shown to increase resistance to collagenase by greater than 3.5 times compared to counterpart afibrillar atelocollagen samples. One-factor ANOVA analysis, with Fisher's LSD post-hoc test, confirms these key findings to be of statistical significance (P < 0.05). The profound physicochemical effects of collagen ultrastructure demonstrated in this study reiterates the need for comprehensive materials disclosure and classification when using these biomaterials.

Interpenetrating polymer networks of collagen, hyaluronic acid, and chondroitin sulfate as scaffolds for brain tissue engineering.

Stem cells can provide neuro-protection and potentially neuro-replacement to patients suffering from traumatic brain injuries (TBI), with a practical option being delivery via engineered scaffolds. Collagen (Coll) and glycosaminoglycan (GAG) have been used as scaffolds for brain tissue engineering yet they often do not support cell differentiation and survival. In this study, we developed interpenetrating polymer network scaffolds comprising Coll, and incorporating two commonly found GAGs in the brain, chondroitin sulfate (CS) and/or hyaluronic acid (HA). We seeded these scaffolds with mouse neural stem cells from the subventricular zone (SVZ) niche. Compared to Coll-alone, all other substrates decreased the percent of nestin+ stem cells. Coll-CS-HA was more efficient at suppressing nestin expression than the other scaffolds; all SVZ cells lost nestin expression within 7 days of culture. In contrast to nestin, the percentage of microtubule associated protein 2 (MAP2+) neurons was greater in scaffolds containing, CS, HA or CS-HA, compared to Coll alone. Finally, Coll-CS increased the percentage of glial fibrillary acidic protein (GFAP+) astrocytes compared to Coll scaffolds. Overall, this work shows that Coll-HA and Coll-CS-HA scaffolds selectively enhance neurogenesis and may be advantageous in tissue engineering therapy for TBI. STATEMENT OF SIGNIFICANCE: Brain injury is devastating yet with few options for repair. Stem cells that reside in the subventricular zone (SVZ) only repair damage inefficiently due to poor control of their cellular progeny and unsuitable extracellular matrix substrates. To solve these problems, we have systematically generated collagen (Coll) scaffolds with interpenetrating polymer networks (IPN) of hyaluronic acid (HA) or chondroitin sulfate proteoglycans (CS) or both. The scaffolds had defined pore sizes, similar mechanical properties and all three stimulated neurogenesis, whereas only CS stimulated astrocyte genesis. Overall, this work suggests that Coll-HA and Coll-CS-HA scaffolds selectively enhance neurogenesis and may be advantageous in tissue engineering therapy for brain repair.

Inflammatory Responses in Oro-Maxillofacial Region Expanded Using Anisotropic Hydrogel Tissue Expander.

OBJECTIVE: Reconstruction of oral and facial defects often necessitate replacement of missing soft tissue. The purpose of tissue expanders is to grow healthy supplementary tissue under a controlled force. This study investigates the inflammatory responses associated with the force generated from the use of anisotropic hydrogel tissue expanders. METHODS: Sprague Dawley rats (n = 7, body weight = 300 g ± 50 g) were grouped randomly into two groups-control (n = 3) and expanded (n = 4). Anisotropic hydrogel tissue expanders were inserted into the frontal maxillofacial region of the rats in the expanded group. The rats were sacrificed, and skin samples were harvested, fixed in formalin, and embedded in paraffin wax for histological investigation. Hematoxylin and eosin staining was performed to detect histological changes between the two groups and to investigate the inflammatory response in the expanded samples. Three inflammatory markers, namely interleukin (IL)-1α, IL-6, and tumor necrosis factor-α (TNF-α), were analyzed by immunohistochemistry. RESULT: IL-1-α expression was only observed in the expanded tissue samples compared to the controls. In contrast, there was no significant difference in IL-6, and TNF-α production. Histological analysis showed the absence of inflammatory response in expanded tissues, and a negative non-significant correlation (Spearman's correlation coefficient) between IL-1-α immune-positive cells and the inflammatory cells (r = -0.500). In conclusion, tissues that are expanded and stabilized using an anisotropic self-inflating hydrogel tissue expander might be useful for tissue replacement and engraftment as the expanded tissue does not show any sign of inflammatory responses. Detection of IL-1-α in the expanded tissues warrants further investigation for its involvement without any visible inflammatory response.

Fabrication of biocompatible porous scaffolds based on hydroxyapatite/collagen/chitosan composite for restoration of defected maxillofacial mandible bone.

Fabrication of scaffolds from biomaterials for restoration of defected mandible bone has attained increased attention due to limited accessibility of natural bone for grafting. Hydroxyapatite (Ha), collagen type 1 (Col1) and chitosan (Cs) are widely used biomaterials which could be fabricated as a scaffold to overcome the paucity of bone substitutes. Here, rabbit Col1, shrimp Cs and bovine Ha were extracted and characterized with respect to physicochemical properties. Following the biocompatibility, degradability and cytotoxicity tests for Ha, Col1 and Cs a hydroxyapatite/collagen/chitosan (Ha·Col1·Cs) scaffold was fabricated using thermally induced phase separation technique. This scaffold was cross-linked with (1) either glutaraldehyde (GTA), (2) de-hydrothermal treatment (DTH), (3) irradiation (IR) and (4) 2-hydroxyethyl methacrylate (HEMA), resulting in four independent types (Ha·Col1·Cs-GTA, Ha·Col1·Cs-IR, Ha·Col1·Cs-DTH and Ha·Col1·Cs-HEMA). The developed composite scaffolds were porous with 3D interconnected fiber microstructure. However, Ha·Col1·Cs-IR and Ha·Col1·Cs-GTA showed better hydrophilicity and biodegradability. All four scaffolds showed desirable blood biocompatibility without cytotoxicity for brine shrimp. In vitro studies in the presence of human amniotic fluid-derived mesenchymal stem cells revealed that Ha·Col1·Cs-IR and Ha·Col1·Cs-DHT scaffolds were non-cytotoxic and compatible for cell attachment, growth and mineralization. Further, grafting of Ha·Col1·Cs-IR and Ha·Col1·Cs-DHT was performed in a surgically created non-load-bearing rabbit maxillofacial mandible defect model. Histological and radiological observations indicated the restoration of defected bone. Ha·Col1·Cs-IR and Ha·Col1·Cs-DHT could be used as an alternative treatment in bone defects and may contribute to further development of scaffolds for bone tissue engineering.

Development of specific collagen scaffolds to support the osteogenic and chondrogenic differentiation of human bone marrow stromal cells.

Type I Collagen matrices of defined porosity, incorporating carbonate substituted hydroxyapatite (HA) crystals, were assessed for their ability to support osteo- and chondrogenic differentiation of human bone marrow stromal cells (HBMSCs). Collagen-HA composite scaffolds supported the osteogenic differentiation of HBMSCs both in vitro and in vivo as demonstrated by histological and micro-CT analyses indicating the extensive penetration of alkaline phosphatase expressing cells and new matrix synthesis with localised areas immunologically positive for osteocalcin. In vivo, extensive new osteoid formation of implant origin was observed in the areas of vasculature. Chondrogenic matrix synthesis was evidenced in the peripheral regions of pure collagen systems by an abundance of Sox9 expressing chondrocytes embedded within a proteoglycan and collagen II rich ECM. The introduction of microchannels to the scaffold architecture was seen to enhance chondrogenesis. Tissue specific gene expression and corresponding matrix synthesis indicate that collagen matrices support the growth and differentiation of HBMSCs and suggest the potential of this platform for understanding the ECM cues necessary for osteogenesis and chondrogenesis.

The impact of critical point drying with liquid carbon dioxide on collagen-hydroxyapatite composite scaffolds.

Collagen-hydroxyapatite composites for bone tissue engineering are usually made by freezing an aqueous dispersion of these components and then freeze-drying. This method creates a foamed matrix which may not be optimum for growing cell colonies larger than a few hundred micrometres due to the limited diffusion of nutrients and oxygen, and the limited removal of waste metabolites. Incorporating a network of microchannels in the interior of the scaffold which may permit the flow of nutrient-rich media has been proposed as a method to overcome these diffusion constraints. A novel three-dimensional printing and critical point drying technique previously used to make collagen scaffolds has been modified to create collagen-hydroxyapatite scaffolds. This study investigates the properties of collagen and collagen-hydroxyapatite scaffolds and whether subjecting collagen and hydroxyapatite to critical point drying with liquid carbon dioxide results in any changes to the individual components. Specifically, the hydroxyapatite component was characterized before and after processing using wavelength-dispersive X-ray spectroscopy, X-ray diffraction and infrared spectroscopy. Critical point drying did not induce elemental, crystallographic or molecular changes in the hydroxyapatite. The quaternary structure of collagen was characterized using transmission electron microscopy and the quarter-staggered array characteristic of native collagen remained after processing. Microstructural characterization of the composites using scanning electron microscopy showed the hydroxyapatite particles were mechanically interlocked in the collagen matrix. The in vitro biological response of MG63 osteogenic cells to the composite scaffolds were characterized using the Alamar Blue, PicoGreen, alkaline phosphate and Live/Dead assays, and revealed that the critical point dried scaffolds were non-cytotoxic.

Encapsulation and release of a hydrophobic drug from hydroxyapatite coated liposomes.

Hydroxyapatite (HA) coated liposomes (HACL) have been successfully manufactured and filled with a model hydrophobic (lipophilic) drug, indomethacin (IMC). These HACL particles have been characterized in terms of particle size and zeta-potential. The liposomes are formed from 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Altering their relative proportions caused the zeta-potential to change from -38.8 to -67.0 mV, with a concomitant change in phase transition temperature from 36.4 to 53.3 degrees C. These changes also affect the drug loading efficiency. The release profiles of IMC have been measured. HA coating of the liposome reduces the release rate of IMC over uncoated liposomes. Under the present experimental conditions 70% of the drug is released after approximately 5h from the liposome, but coating with HA changes this time to over 20 h. Perhaps most importantly, it has been observed that for uncoated liposomes, IMC is released at a greater rate at pH=7.4 than at pH=4. However, coating with HA reduced the rate at pH=7.4 compared to pH=4. This behaviour arises because IMC is more soluble under basic conditions, but HA is more soluble under acidic conditions. This behaviour shows that it is now possible to have environmental control over the release of drugs from HA-coated liposomes.

Interaction of human valve interstitial cells with collagen matrices manufactured using rapid prototyping.

Rapid prototyping is a novel process for the production of scaffolds of predetermined size and three-dimensional shape. The aim of the study was to determine the feasibility of this technology for producing scaffolds for tissue engineering an aortic valve and the optimal concentration of collagen processed in this manner that would maintain viability and promote proliferation of human valve interstitial cells. Scaffolds of 1%, 2% and 5% w/v bovine type-I collagen were manufactured using rapid prototyping. Valve interstitial cells isolated from three human aortic valves were seeded on the scaffolds and cultured for up to 4 weeks. Cell viability was assessed using the CellTiter 96 Aq(ueous) One Solution Cell Proliferation Assay and cell death by lactate dehydrogenase (LDH) measurement. Valve interstitial cells remained viable and proliferated within the collagen scaffolds. Cells consistently proliferated to a greater extent on 1% collagen scaffolds rather than either 2% or 5% collagen and after 4 weeks reached 212+/-33.1%, 139+/-25.9% and 129+/-38.3% (mean+/-SD) of their initial seeding density on 1%, 2% and 5% collagen scaffolds, respectively. LDH analysis demonstrated that there was minimal cell death indicating that the collagen scaffold was not toxic to human valve interstitial cells. Rapid prototyping provides a route to optimize biological scaffold designs for tissue engineering cardiac valves. This technology has the versatility to create scaffolds that are compatible with the specific needs of the valve interstitial cells and should enhance cell viability, proliferation and function.

Collagen scaffolds reinforced with biomimetic composite nano-sized carbonate-substituted hydroxyapatite crystals and shaped by rapid prototyping to contain internal microchannels.

The next generation of tissue engineering scaffolds will be made to accommodate blood vessels and nutrient channels to support cell survival deep in the interior of the scaffolds. To this end, we have developed a method that incorporates microchannels to permit the flow of nutrient-rich media through collagen-based scaffolds. The scaffold matrix comprises nano-sized carbonate-substituted hydroxyapatite (HA) crystals internally precipitated in collagen fibers. The scaffold therefore mimics many of the features found in bone. A biomimetic precipitation technique is used whereby a collagen membrane separates reservoirs of calcium and phosphate solutions. The collision of calcium and phosphate ions diffusing from opposite directions results in the precipitation of mineral within the collagen membrane. Transmission electron microscopy analysis showed the dimension of the mineral crystals to be approximately 180 x 80 x 20 nm, indicating that the crystals reside in the intermicrofibril gaps. Electron diffraction indicated that the mineral was in the HA phase, and infrared spectroscopy confirmed type A carbonate substitution. The collagen-HA membrane is then used to make 3-dimensional (3D) scaffolds: the membrane is shredded and mixed in an aqueous-based collagen dispersion and processed using the critical point drying method. Adjusting the pH of the dispersion to 5.0 before mixing the composite component preserved the nano-sized carbonate-substituted HA crystals. Branching and interconnecting microchannels in the interior of the scaffolds are made with a sacrificial mold manufactured by using a 3D wax printer. The 3D wax printer has been modified to print the mold from biocompatible materials. Appropriately sized microchannels within collagen-HA scaffolds brings us closer to fulfilling the mass transport requirements for osteogenic cells living deep within the scaffold.

Potential for synthesis and degradation of extracellular matrix proteins by valve interstitial cells seeded onto collagen scaffolds.

Matrix remodeling, which involves proteolytic enzymes, such as the matrix metalloproteinases (MMPs), is of significant importance with respect to tissue engineering a heart valve construct. The ability of valve interstitial cells (ICs) to release these enzymes in biological scaffolds and to synthesize their own matrix has not been adequately studied, and this has important implications for tissue engineering. Cultured human aortic valve ICs were seeded onto a 3-dimensional type I collagen matrix for 28 days, whereby the presence of the remodeling enzymes, MMPs, were determined using immunohistochemistry, and detection of extracellular matrix (ECM) gene expression was performed using in situ hybridization. The collagenases, stromelysins, and membrane-type MMPs were expressed in 1%, 2%, and 5% collagen scaffolds after 28 days, whereas gelatinase expression was not observed. In situ hybridization revealed the presence of the ECM messenger ribonucleic acid (mRNA) in cells cultured in collagen scaffolds however, an increase in all three mRNAs was only detected in the 1% collagen scaffolds. The presence of collagenases, stromelysins, and membrane-type MMPs indicate that human valve ICs have the capacity to remodel type I collagen scaffold and that the genes necessary for synthesizing matrix have been turned on within the cells themselves. Scaffold composition also demonstrated differential effects onMMPexpression. These observations are of relevance with respect to the development of tissue-engineered heart valves.

Magnetic resonance imaging of ferumoxide-labeled mesenchymal stem cells seeded on collagen scaffolds-relevance to tissue engineering.

Mesenchymal stem cells (MSCs) are a promising candidate cell for tissue engineering. Magnetic resonance imaging (MRI) has been proven effective in visualizing iron-labeled stem cells; however, the efficiency of this approach for visualization of cells seeded on scaffolds intended for use as tissue-engineered heart valves has not been assessed. MSCs were labeled by incubating for 48 h with ferumoxide and poly-L-lysine as transfecting agent. Any detrimental effect of iron labeling on cell viability, proliferation, and differentiation was examined using appropriate functional assays. Change in the nuclear magnetic relaxation properties of labeled cells was determined using in vitro relaxometry of cells seeded in 3-dimensional collagen gels. Images of labeled and non-labeled cells seeded onto 1% type I bovine collagen scaffolds were obtained using MRI. The presence of intracellular iron in labeled cells was demonstrated using Prussian blue staining, confocal microscopy, and electron microscopy. Cell viability, proliferation, and differentiation were comparable in labeled and non-labeled cells. The T2 relaxation time was 40% to 50% shorter in ferumoxide-labeled cells. Labeled cells seeded on scaffolds appeared as areas of reduced signal intensity in T2 weighted images. Ferumoxide labeling persisted and remained effective even on scans performed 4 weeks after the labeling procedure. Ferumoxide labeling of human MSCs seeded on collagen scaffolds is an effective, non-toxic technique for visualization of these cells using MRI. This technique appears promising for cell tracking in future tissue-engineering applications.

The Mechanical, Structural, and Compositional Changes of Tendon Exposed to Elastase.

The mechanical response of tendon is dependent on the interaction of structural molecules that constitute the extracellular matrix. However, little is known about the role of elastic fibers that are present in this structure. Elastase treatments have been used to elucidate the mechanical role of elastic fibers in numerous tissues. Here, we show that a standard elastase treatment affects the mechanical properties of tendon, including the ultimate tensile strength and failure strain. Moreover, elastase-treated specimens exhibit significant structural and compositional changes including crimp undulation and release of glycosaminoglycans. These data demonstrate that a common elastase treatment has a complex digestion profile that influences the structure-function relationship of tendon. Thus, defining the mechanical role of elastic fibers in tendon using this technique is challenging. This introduces new and exciting questions regarding the function of elastic fibers in tendon, which may not be as well understood as previously thought.

Development of a novel anisotropic self-inflating tissue expander: in vivo submucoperiosteal performance in the porcine hard palate.

BACKGROUND: The advent of self-inflating hydrogel tissue expanders heralded a significant advance in the reconstructive potential of this technique. Their use, however, is limited by their uncontrolled isotropic (i.e., uniform in all directions) expansion. METHODS: Anisotropy (i.e., directional dependence) was achieved by annealing a hydrogel copolymer of poly(methyl methacrylate-co-vinyl pyrrolidone) under a compressive load for a specified time period. The expansion ratio is dictated by the percentage of vinyl pyrrolidone content and the degree of compression. The expansion rate is modified by incorporating the polymer within a silicone membrane. The in vivo efficacy of differing prototype devices was investigated in juvenile pigs under United Kingdom Home Office Licence. The devices were implanted within a submucoperiosteal pocket in a total of six porcine palates; all were euthanized by 6 weeks after implantation. A longitudinal volumetric assessment of the expanded tissue was conducted, in addition to postmortem analysis of the bony and mucoperiosteal palatal elements. RESULTS: Uncoated devices caused excessive soft-tissue expansion that resulted in mucoperiosteal ulceration, thus necessitating animal euthanasia. The silicone-coated devices produced controlled soft-tissue expansion over the 6-week study period. There was a statistically significant increase in the volume of expanded soft tissue with no evidence of a significant acute inflammatory response to the implant, although peri-implant capsule formation was observed. Attenuation of the bony palatal shelf was noted. CONCLUSION: A unique anisotropic hydrogel device capable of controlled expansion has been developed that addresses a number of the shortcomings of the technology hitherto available.

Extracellular matrix production by adipose-derived stem cells: implications for heart valve tissue engineering.

A key challenge in tissue engineering a heart valve is to reproduce the major tissue structures responsible for native valve function. Here we evaluated human adipose-derived stem cells (ADSCs) as a source of cells for heart valve tissue engineering investigating their ability to synthesize and process collagen and elastin. ADSCs were compared with human bone marrow mesenchymal stem cells (BmMSCs) and human aortic valve interstitial cells (hVICs). ADSCs and BmMSCs were stretched at 14% for 3 days and collagen synthesis determined by [(3)H]-proline incorporation. Collagen and elastin crosslinking was assessed by measuring pyridinoline and desmosine respectively, using liquid chromatography/mass spectrometry. Three-dimensional culture was obtained by seeding cells onto bovine collagen type I scaffolds for 2-20 days. Expression of matrix proteins and processing enzymes was assessed by Real Time-PCR, immunofluorescence and transmission electron microscopy. Stretch increased the incorporation of [(3)H]-proline in ADSCs and BmMSCs, however only ADSCs and hVICs upregulated COL3A1 gene. ADSCs produced collagen and elastin crosslinks. ADSCs uniformly populated collagen scaffolds after 2 days, and fibrillar-like collagen was detected after 20 days. ADSCs sense mechanical stimulation and produce and process collagen and elastin. These novel findings have important implications for the use of these cells in tissue engineering.

Controlled release of amoxicillin from hydroxyapatite-coated poly(lactic-co-glycolic acid) microspheres.

Negatively charged poly(lactic-co-glycolic acid) (PLGA) microspheres with an encapsulated hydrophilic antibiotic (amoxicillin) have been prepared by the solid-in-oil-in-water (s/o/w) method using the anionic surfactant, sodium dodecyl sulfate (SDS). Drug encapsulation efficiency is over 40%. Successful coating of hydroxyapatite (HA) on these negatively charged PLGA microspheres has been achieved by a dual constant composition method in 3-6 h. The HA-coated PLGA microspheres (HPLG) have been characterised by zeta-potential and particle size measurements and the coating has been confirmed to be calcium deficient HA by analysis of X-ray diffraction, Fourier transform infrared spectroscopy and wavelength dispersive spectroscopy. The morphology of HPLG was studied by scanning electron microscopy, and cross sections of HPLG microspheres were prepared and imaged using focused ion beam microscopy. In-vitro drug release experiments in PBS (pH7.4) showed a sustained release profile for at least 31 days with little initial burst release. It shows a triphasic drug release profile commonly observed for biodegradable polymers.

Controlling the processing of collagen-hydroxyapatite scaffolds for bone tissue engineering.

Scaffolds are an important aspect of the tissue engineering approach to tissue regeneration. This study shows that it is possible to manufacture scaffolds from type I collagen with or without hydroxyapatite (HA) by critical point drying. The mean pore sizes of the scaffolds can be altered from 44 to 135 microm depending on the precise processing conditions. Such pore sizes span the range that is likely to be required for specific cells. The mechanical properties of the scaffolds have been measured and behave as expected of foam structures. The degradation rate of the scaffolds by collagenase is independent of pore size. Dehydrothermal treatment (DHT), a common method of physically crosslinking collagen, was found to denature the collagen at a temperature of 120 degrees C resulting in a decrease in the scaffold's resistance to collagenase. Hybrid scaffold structures have also been manufactured, which have the potential to be used in the generation of multi-tissue interfaces. Microchannels are neatly incorporated via an indirect solid freeform fabrication (SFF) process, which could aid in reducing the different constraints commonly observed with other scaffolds.